Lineage reconstruction

Reconstruction of an Ig lineage requires the following steps:

  1. Load a Change-O tab-delimited database file and select a clone
  2. Preprocess the clone to remove gap characters and duplicate sequences
  3. Run PHYLIP, parse the output, and modify the tree topology

Example data

A small example Change-O database, ExampleDb, is included in the alakazam package. Lineage reconstruction requires the following fields (columns) to be present in the Change-O file:

  • V_CALL
  • J_CALL
# Load required packages

# Select clone from example database
sub_db <- subset(ExampleDb, CLONE == 3138)

Preprocess a clone

Before a lineage can be constructed the sequences must first be cleaned of gap (-, .) characters added by IMGT, duplicate sequences must be removed, and annotations must be combined for each cluster of duplicate sequences. Optionally, “ragged” ends of sequences, such as may occur from primer template switching, may also be cleaned by masking mismatched positions and the leading and trailing ends of each sequence. The function makeChangeoClone is a wrapper function which combines these steps and returns a ChangeoClone object which may then be passed into the lineage reconstruction function.

Two arguments to makeChangeoClone control which annotations are retained following duplicate removal. Unique values appearing within columns given by the text_fields arguments will be concatenated into a single string delimited by a “,” character. Values appearing within columns given by the num_fields arguments will be summed.

# This example data set does not have ragged ends
# Preprocess clone without ragged end masking (default)
clone <- makeChangeoClone(sub_db, text_fields=c("SAMPLE", "ISOTYPE"), 

# Show combined annotations
clone@data[, c("SAMPLE", "ISOTYPE", "DUPCOUNT")]
## 1    +7d     IgA        1
## 2    +7d     IgG        1
## 3    +7d IgA,IgG       10
## 4    +7d     IgG       36
## 5    +7d     IgA       10
## 6    +7d     IgG       13


Lineage construction uses the dnapars (maximum parsimony) application of the PHYLIP package. The function buildPhylipLineage performs a number of steps to execute dnapars, parse its output, and modify the tree topology to meet the criteria of an Ig lineage. This function takes as input a ChangeoClone object output by makeChangeoClone and returns an igraph graph object. The igraph graph object will contain clone annotations as graph attributes, sequence annotations as vertex attributes, and mutations along edges as edge attributes.

The system call to dnapars requires a temporary folder to store input and output. This is created in the system temporary location (according to base::tempfile), and is not deleted by default (only because automatically deleting files is somewhat rude). In most cases, you will want to set rm_temp=TRUE to delete this folder.

# Run PHYLIP and parse output
dnapars_exec <- "~/apps/phylip-3.69/dnapars"
graph <- buildPhylipLineage(clone, dnapars_exec, rm_temp=TRUE)
# The graph has shared annotations for the clone
## 1  3138              60 IGHV3-49  IGHJ5
# The vertices have sequence specific annotations
## 1 GN5SHBT06HH3QD     IgA       10
## 2 GN5SHBT08F45HV IgA,IgG       10
## 3       Germline    <NA>       NA
## 4 GN5SHBT06IFV0R     IgG       13
## 5 GN5SHBT08I3P11     IgG       36
## 6 GN5SHBT01BXJY7     IgG        1
## 7 GN5SHBT01EGEU6     IgA        1

Plotting of the lineage tree

Plotting of a lineage tree may be done using the built-in functions of the igraph package. The default edge and vertex labels are edge weights and sequence identifiers, respectively.

# Plot graph with defaults

plot of chunk Lineage-Vignette-6

The default layout and attributes are not very pretty. We can modify the graphical parameter in the usual igraph ways. A tree layout can be built using the layout_as_tree layout with assignment of the root position to the germline sequence, which is named “Germline” in the object returned by buildPhylipLineage.

# Modify graph and plot attributes
V(graph)$color <- "steelblue"
V(graph)$color[V(graph)$name == "Germline"] <- "black"
V(graph)$color[grepl("Inferred", V(graph)$name)] <- "white"
V(graph)$label <- V(graph)$ISOTYPE
E(graph)$label <- ""

# Remove large default margins
par(mar=c(0, 0, 0, 0) + 0.1)
# Plot graph
plot(graph, layout=layout_as_tree, edge.arrow.mode=0, vertex.frame.color="black",
     vertex.label.color="black", vertex.size=40)
# Add legend
legend("topleft", c("Germline", "Inferred", "Sample"), 
       fill=c("black", "white", "steelblue"), cex=0.75)

plot of chunk Lineage-Vignette-7

Which is much better.

Batch processing lineage trees

Multiple lineage trees may be generated at once, by splitting the Change-O data.frame on the clone column.

# Preprocess clones
clones <- ExampleDb %>%
    group_by(CLONE) %>%
    do(CHANGEO=makeChangeoClone(., text_fields=c("SAMPLE", "ISOTYPE"), 
# Build lineages
dnapars_exec <- "~/apps/phylip-3.69/dnapars"
graphs <- lapply(clones$CHANGEO, buildPhylipLineage, 
                 dnapars_exec=dnapars_exec, rm_temp=TRUE)
# Note, clones with only a single sequence will not be processed.
# A warning will be generated and NULL will be returned by buildPhylipLineage
# These entries may be removed for clarity
graphs[sapply(graphs, is.null)] <- NULL

# The set of tree may then be subset by node count for further 
# analysis, if desired.
graphs <- graphs[sapply(graphs, vcount) >= 5]

Converting between graph, phylo, and newick formats

While much of analysis in alakazam focuses on using igraph graph objects, R phylo objects are capable of being used by a rich set of phylogenetic analysis tools in R. Further, stand-alone phylogenetics programs typically import and export trees in Newick format.

To convert to trees in graph format to phylo format, use graphToPhylo. These objects can now be used by functions detailed in other R phylogenetics packages such as ape. To export lineage trees as a Newick file, use the write.tree function provided in ape.

# Modify graph and plot attributes
V(graph)$color <- categorical_pal(8)[1]
V(graph)$label <- V(graph)$name
E(graph)$label <- E(graph)$weight
##plot lineage tree using igraph
plot(graph, layout=layout_as_tree)

plot of chunk Lineage-Vignette-13

# convert to phylo
phylo <- graphToPhylo(graph)

#plot using ape
plot(phylo, show.node.label=TRUE)

plot of chunk Lineage-Vignette-13

#write tree file in Newick format
ape::write.tree(phylo, file="example.tree")

To import lineage trees as phylo objects from Newick files, use the read.tree function provided in the ape package. To convert this phylo object to a graph object, use the phyloToGraph function with the germline sequence ID specified using the germline option. Note that while some of the nodes in more complex trees may rotate during this process, their topological relationships will remain the same.

#read in tree as phylo object
phylo_r <- ape::read.tree("example.tree")

#convert to graph object
graph_r <- phyloToGraph(phylo_r, germline="Germline")

#plot converted form using igraph - it's the same as before

plot of chunk Lineage-Vignette-14